Primer design and in silico analysis using CLUSTALW and MUSCLE for L-arabinose isomerase (araA) gene detection in thermophilic bacteria

Low-calorie sweetener, D-tagatose, has sweet characteristic comparable to sucrose. The use of D-tagatose has been deemed as safe, hence D-tagatose is categorized as GRAS material. D-tagatose can be synthesized through isomerization catalyzed by thermostable L-arabinose isomerase encoded by araA gene. Thermostable L-AI can be isolated from thermophilic bacteria, especially from hot spring water source in Sikidang, Dieng Plateau. Determination of capability of bacterial isolates to produce thermostable L-AI can be conducted through gene detection. The method of gene detection involves PCR using a specifically designed primer to amplify the targeted gene. Here, the study was aimed to design primers which amplify specific target within araA gene of partially characterized bacterial strain. The steps involved data mining through NCBI, multiple sequence alignment using CLUSTALW and MUSCLE, primer generation and candidates sorting, in silico PCR and primer characteristics checking by using OligoCalc. The result showed that in the context of this study, CLUSTALW and MUSCLE works in conjunction with each other. There are 4 pairs of primers which could be deduced to amplify the targeted gene. In silico PCR showed that primers iaraAF1 and iaraAR3 are specifically proven to amplify the targeted gene from Bacillus group.